We have developed a soluble, cell-free system that carries out the replication of exogenous DNA templates containing the SV40 origin of DNA replication. We plan to use this system to explore the molecular mechanisms involved in viral and cellular DNA replication. Our specific aims for the initial grant period are as follows: (1) To analyze the structure and function of the SV40 origin of replication by studying the effects of deletions and point mutations on DNA replication in vitro. (2) To purify and functionally characterize cellular proteins involved in DNA replication with particular emphasis on the proteins required for the initiation reaction. (3) To extend the in vitro system to the analysis of initiation at cellular origins of replication.